From geology to biology: an interdisciplinary course in crystal growth

The authors share experience of teaching an interdisciplinary university course in crystal growth with examples ranging from geology to biology.

Semiconductors with p-and n-type of conductivity.
Photo-and thermoluminescent materials with impurity luminescence. Phase diagrams 'diopside -anorthite' and 'k-feldspar -SiO2' as models of crystallization of silicate melt. Liquidus phase. Restite (eutectic) melt and its textural signs. The transition from idiomorphic to xenomorphic crystals during the crystallization.
The conditions of crystallization in deep magmatic chamber and erupted lava. Cooling rates, temperature gradients, oversaturation. The variation of the rate of crystal nucleation and growth depending on oversaturation. Multistage crystal growth in basaltic melts during its ascent and effusion. The resulting textures of intrusive (granite) and effusive (basalt) rocks.
Co-crystallization from eutectic melt: oriented (graphic) intergrowths of minerals -pegmatites. The necessary conditions (over-cooling, diffusion and crystal growth rates, the presence of water) for their growth. Part 2. Solid state growth in the Earth's crust Exsolution. Solid solution. Isomorphic elements. Ordering of solid solution, its crystal chemical prerequisites. Exsolution and its thermodynamic interpretation. Solvus, immiscibility range. Exsolution processes in minerals; cooling rates and the degree of separation of the product phases. Homogeneous and heterogeneous nucleation. Energetic parameters of the nucleus surface: elastic strain and surface energy. Equilibrium (chemical) and coherent solvus.
Metamorphic reactions. Metamorphic processes in the Earth's crust, pressure and temperature as their principal factors.
Lithostatic and tectonic pressure.
Polymorphic transition. Polymorphism in Al2SiO5 system. Impact metamorphism, its P-T parameters. Popigai astrobleme. Impact diamondspseudomorphs after graphite. Lonsdeilite (hexagonal diamond) as a sign of impact origin of diamond. Fine structure of lonsdeilite-diamond intergrowths.
Methods of obtaining biopolymers.
Molecular mass and the number of the amino acid residues. Examples of an insulin monomer, lysozyme, influenza virus glycoprotein, eukaryotic small ribosomal subunit (40S). The sizes of hemoglobin and erythrocyte size.
Requirements to a solution of a biopolymer suitable for crystallization.
A general overview of the methods of structure determination of biopolymers with analysis of the Protein Database (PDB) statistics on the number of structures defined by each method.
The main stages of the crystallization of the biopolymers.
Concepts: the region of the undersaturation, solubility curve, metastable zone, nucleation zone, precipitation zone, the supersaturation region, phase diagrams for the systems where supersaturation is achieved by using an antisolvent.
Methods of the crystallization of biopolymers and different routes of reaching nucleation: Vapor-diffusion method (hanging drop, sitting drop, sandwich drop), batch experiments, dialysis, Free interface diffusion method. The method of counter-diffusion.
Crystallization of membrane proteins.
Equipment for preparing protein solutions, micro-and nano-droplets, crystal growth at a constant desirable temperature.
following concepts are discussed in relation to this laboratory exercise: seed, saturated solution, faceting, habit.
2. Crystal growth To obtain the best defect-free octahedral crystals, it is necessary to choose the most transparent and well-faceted seed crystals, without internal defects or inclusions (this can be achieved using an optical microscope). The selected crystal is fixed on a thread tied to a stick (toothpick, pencil, etc.) and placed into a saturated solution of potassium alum (Fig. S1a). The container for crystallization is covered with filter paper and left to grow a larger crystal. It is necessary to periodically (once every 3-4 days) check the growth of the crystal to avoid pollution of its surface with small crystals. The appearance of additional seed crystals on the thread and at the bottom of the crystallization container must also be avoided. If necessary, the thread (or the crystal surface) should be cleaned mechanically or using a hot tap water jet (without harming the target crystal). It is also necessary to check the level of the mother liquor in the container and, if it has decreased due to evaporation, top-up the beaker from the previously prepared saturated solution of potassium alum (from Step 1). After extraction from the solution and drying (Fig. 2a, Fig. S1b,c), the crystals must be stored in a sealed container to prevent dehydration, since alum crystallizes as a crystal hydrate.

Cutting of a spherical crystal and its growth A large crystal grown at
Step 2 is evenly sanded with sandpaper until it becomes spherical. The spherical crystal is suspended in a saturated solution of potassium alum under isothermal conditions to observe the growth figures of the faces on the surface of the originally spherical sample (2-4 weeks).
Finishing the lab work: wash all the glassware, discuss the obtained results.

6b. Crystallization of L-ascorbic acid (vitamin C) -L-serine co-crystals by slow evaporation
Main goal -crystal growth of co-crystals of L-ascorbic acid with L-serine.
Description of lab work: Dissolve 105 mg of L-serine and 176 mg of L-ascorbic acid in 600 ml of water in plastic test tubes. Dissolution takes 10-15 minutes. Cover a glass microscope slide with Parafilm® M, so that part of the glass remains uncovered. The aqueous solution is filtered with a syringe through cotton wool or filter nozzle into a new plastic test tube. A droplet of 50 µl is placed on the glass surface and 3 bigger droplets of ca. 100 µl, 150 µl and 300 µl are put on the surface covered by Parafilm® M. Attention should be paid to the droplet shape. When placed on the glass surface the drop tends to lose its shape. Instead, when a drop is placed on the hydrophobic Parafilm® M surface, the drops maintain an arched shape. The small drop of 50 µl on the glass is used to obtain crystal nuclei that will be taken to grow bigger crystals in drops places on Parafilm® M (see Fig. S2). The following processes and concepts are discussed: solubility curve for a two-component system, change in the concentration of a dissolved compound during solvent evaporation, "metastable zone", nucleation, supersaturation, spontaneous crystallization, co-crystal.
After approximately 40 min from the beginning of the experiment, crystal nuclei from the droplet on the glass surface are transferred using a metal needle into all 3 drops of different volumes (100, 150, 300 µl) located on the Parafilm® M (see Fig. S2). Crystals should begin to grow in the 100 µl droplet, and dissolve in the larger droplets if the humidity is high.
Observe the growth of crystals in the 100 µl drop directly through a microscope. After a while, put the nuclei crystals again into drops of 150 µl and 300 µl again. Observe crystal growth or dissolution of the nuclei crystals again. As soon as it is possible to grow crystals larger than 0.2 mm, the crystals are extracted from the drop. Select and preserve the single crystals in cryo-oil for further testing on a single crystal X-ray diffractometer.
Finishing the lab work: wash all the glassware, turn off all equipment, discuss the experience gained, main points of theory and the obtained results.

6с. Lysozyme
Main goal -to get some experience with the crystallization of biopolymers Approximate time limit: 2 academic hours Tasks: falcon tubes, 1.5 ml Eppendorf tubes, racks for Eppendorf tubes, pH meter, balances, laboratory fridge, microscope with polarizer and analyzer, WACKER® SILICON PASTE P4 or analog.

Description of lab work
Procedures carried out by an assistant before the class starts. It is also possible to have this work performed by the most advanced students: 15 mg of lysozyme is placed into a 1.5 ml Eppendorf tube, and dissolved in 485 µl of distilled water, thus making a 30 mg/ml lysozyme solution. Lysozyme solutions with concentrations of 50 mg/ml and 70 mg/ml should be also prepared. A 1 M NaAc/Hac stock buffer with pH = 4 is prepared by slowly adding 1 M NaAc solution to a 1 M Hac solution while stirring to reach pH = 4, ensuring that the pH is controlled during this process. The 4 M stock solutions of NaCl are prepared by dissolving in distilled water the corresponding amount of NaCl in a 500 ml volumetric flask. The lysozyme solutions are stored in the fridge.
Work done by students: Students are divided into pairs and calculate the required volumes of 4 M NaCl stock solutions and 1 M NaAc/Hac buffer to get 2 ml precipitant solutions (i.e. NaCl in buffer solution) with NaCl concentrations of 1.1M, 1.2M, 1.3M, and 1.4M. A teacher checks the calculated ratios and then each student prepares the 2 ml solutions using P1000-type micropipettes. The teacher shows how to grease the edges of the crystallization vessel, and each student repeats this procedure.
A teacher and the students then fill the wells of a 24-well crystallization plate with the antisolvent solutions (500 µl), so that wells А1-A3 are filled with the 1.1 M NaCl solution, wells B1-B3 are filled with 1.2 M NaCl solution, wells C1-C3 are filled with 1.3 M NaCl solution, and wells D1-D3 are filled with the 1.4 M NaCl solution (Fig. S3). Each time before changing the antisolvent solution, the nozzle of the pipette must be changed.
A 2 µl droplet of the 30 mg/ml lysozyme solution is then put on the siliconized glass using a P10-type micropipette; the tip of the micropipette is removed afterwards (Fig.S4a). After that, 2 µl of the antisolvent solution from well A1 are added to the lysozyme droplet (Fig.S4b). The glass with a 4µl droplet is turned over and pasted above the antisolvent solution placed in the A1 well using grease (Fig.S4c). This is first demonstrated by a teacher, and then reproduced by the students. As a result, droplets of the 15 mg/ml lysozyme (initial concentration of the 30 mg/ml twice diluted) solution hang above wells А1, В1, С1, D1 (Fig.S4b). The same sequence is carried out with lysozyme solutions of 50 and 70 mg/ml and then placed above A2-D2 and A3-D3 wells, respectively (Fig. S3).
Above these antisolvent solutions the lysozyme crystals grow in approximately 40 mins. Students should be warned that the vapor diffusion process does not occur in such a short time and crystallization is due to the amount of precipitator solution that was added to the drop. In the case of other proteins (not lysozyme) crystallization experiment takes from several days to weeks.
The crystals are inspected under an optical microscope in non-polarized and polarized light.
The number and quality of crystals obtained under different crystallization conditions are compared and discussed.